Heterochromatin comprises tightly compacted repetitive regions of eukaryotic chromosomes. It is inherited through mitosis and has roles in transcriptional silencing, centromere specification and genome integrity, which profoundly impact epigenetic mechanisms in human health and disease. We have found that the epigenetic inheritance of heterochromatin in fission yeast requires RNA interference (RNAi) to guide histone modification, which occurs during the DNA replication phase of the cell cycle. In the fission yeast S.pombe centromeric repeats have an alternating arrangement of small RNA clusters and origins of replication that makes collision of the transcription and replication machineries all but inevitable. We propose that RNA interference promotes release of RNA polymerase (PolII) during S phase, allowing completion of centromeric DNA replication by the leading strand DNA polymerase. DNA Polymerase epsilon directly recruits the histone-modifying Rik1 complex and so can spread heterochromatin along with DNA replication. In the absence of RNAi, stalled forks are repaired by homologous recombination (HR) without histone modification, so that HR is essential in the absence of RNAi. This model may explain the participation of non-coding RNA and DNA replication in many examples of epigenetic silencing, including paramutation in plants, and imprinting and X-inactivation in mammals. S.pombe is an outstanding model system for cell cycle research, heterochromatic silencing, and RNAi. We will examine the roles of DNA replication, RNA Polymerase release, DNA recombination and repair in heterochromatic histone modification mediated by RNAi. We will utilize models of heterochromatic nucleation and RNAi, as well as chromosome profiling and genetic analysis, to test our hypothesis. We will build on our recent results concerning the roles of the Rik1 complex and Centromere-binding protein B in DNA replication and repair, as well as RNA interference. PUBLIC HEALTH RELEVANCE: Epigenetic mechanisms alter gene function independent of DNA sequence, and have profound effects on health and disease. RNA interference impacts these mechanisms by guiding the modification of histones associated with the DNA, ensuring specificity and avoiding inappropriate gene silencing. We have found that replication of the chromosome during cell division occurs at the same time as RNA interference, and that these mechanisms interact to cause silencing. The key molecules involved are conserved from yeast to humans, and are implicated in childhood disease, mental retardation, aging and cancer. Our findings suggest that therapies that target these molecules may also impact gene expression and chromosome organization. We will investigate the underlying mechanism to determine the causes and consequences of RNAi- mediated modification of chromatin during the DNA replication phase of the cell cycle.